GENERAL WETLAND SCIENCE

The Tensile Root Strength of Spartina patens: Responseto Atrazine Exposure and Nutrient Addition

Lauris O. Hollis1 & R. Eugene Turner1

Received: 26 July 2018 /Accepted: 14 January 2019 /Published online: 31 January 2019# The Author(s) 2019

AbstractCoastal wetlands are receiving basins for inland runoff that contains numerous compounds such as nutrients and herbicides,which may have negative effects on wetland plants. Spartina patens is a dominant emergent macrophyte in low salinity wetlandswhose biomechanical properties contribute to wetland stability against erosive forces and herbivore grazing. We conducted twogreenhouse experiments with six levels of nutrients and three levels of atrazine doses to test the hypothesis that exposure tonutrients and atrazine changes the tensile root strength of S. patens. The results revealed that the tensile root strength of S. patenswas not affected by either atrazine exposure or nutrient addition after 60 days, whereas the plants treated with atrazine, nutrientaddition, or an atrazine-nutrient combination had significantly less tensile root strength than the Control after 212 days. Therewere no significant differences in tensile root strength between the main effects and treatment combinations, and hence, nointeractive effects of nutrient addition and atrazine exposure. These results suggest that the influx of poor quality water intocoastal wetlands will decrease the tensile root strength of S. patens and make coastal wetlands even more vulnerable to sea levelrise and climate change.

Keywords Tensile root strength . Spartina patens . Roots .Wetlands . Atrazine . Nutrient addition . Nitrogen . Phosphorus

Introduction

Wetlands may be receiving basins for surface and subsurfaceflow because of their hydrogeomorphic position in the land-scape. The hydropattern of these hydrologic inputs can influ-ence the water quality and biogeochemical processes in wet-lands and adjacent ecosystems. Nonpoint pollution sources, inparticular, bring nutrients and herbicides into wetlands as aconsequence of land use and the increased use of reactivenitrogen and phosphorus by agricultural operations to producefood, fuel, and fiber for human benefit (Galloway et al. 2008;Rabalais 2009; Ruddiman 2013).

These anthropogenic sources of nutrients may increase eu-trophication frequency and severity (Nixon 1995), which maycreate hypoxic or ‘dead zones’ in marine or estuarine

environments (Turner et al. 2008; Rabalais 2009), alter nutri-ent cycles (Justić et al. 1995), and disrupt the trophic dynamicsin food webs (Reish et al. 1980; Conley et al. 1993; Turneret al. 1998). This increased influx of nutrients provides nu-merous alternate electron acceptors for oxidation-reduction(hereafter, redox) reactions, which are utilized by microbialorganisms. Carbon, for example, acts as the electron donorin these reactions, and phosphorus additions may result inthe loss of plant biomass (Darby and Turner 2008a). It is theloss of biomass, particularly the belowground biomass, whichhas been implicated in the degradation of coastal marshes.Excess nutrient loads can degrade the belowground biomassof wetland plants (Darby and Turner 2008a, b; Wigand et al.2009; Deegan et al. 2012; Wigand et al. 2014; Bodker et al.2015), which may result in reduced soil strength (Turner2011). Thus, anthropogenic inputs may reduce the ability ofcoastal wetlands to maintain soil elevation and keep pace withsea level rise.

Herbicides are also introduced with agricultural fertilizeruse. The herbicide atrazine (6-chloro-N-ethyl-N-(1-methylethyl)-1,3,5-triazine-2,4-diamine) is used for pre-emergence and post-emergent control of broadleaf plantsand grasses in agricultural and forestry operations (Ghoshand Philip 2006). Atrazine binds with a protein complex in

* Lauris O. Hollislholli8@lsu.edu

R. Eugene Turnereuturne@lsu.edu

1 Department of Oceanography and Coastal Sciences, Louisiana StateUniversity, 1195 Energy Coast and Environment Building, BatonRouge, LA 70803, USA

Wetlands (2019) 39:759–775https://doi.org/10.1007/s13157-019-01126-1

Photosystem II in plant chloroplasts and inhibits the transfer ofelectrons, which, in turn, disrupts the formation and release ofoxygen (USEPA 2016). The herbicide is absorbed by plantroots and translocated through the xylem to the leaves andapical meristem, where chlorosis and death are caused byinhibition of photosynthesis due to blockage of the transportof electrons to Photosystem I (Donnelly et al. 1993; Cejudo-Espinosa et al. 2009). Atrazine may also undergo transforma-tion in the soil, soil porewater, and water column into its pri-mary metabolites deethylatrazine (DEA), deisopropylatrazine(DIA), and hydroxyatrazine (HA) (Clay and Koskinen 1990a,b; Seybold and Mersie 1996; Mersie et al. 1998). These me-tabolites may be further transformed along a degradation path-way to form cyanuric acid and then biuret by cleavage of thering structure via hydrolysis (Kruger et al. 1993a, b). The endproducts of atrazine degradation are carbon dioxide and ammo-nia. Consequently, atrazine may be a potential source of addi-tional nitrogen input to wetland macrophytes via the processesof ammonification, which converts ammonia to ammonium,and nitrification, which then oxidizes ammonium and convertsit to nitrate.

The effects of atrazine on agricultural crops are wellknown, but there is a lack of consensus about how atrazineaffects wetland plants. For example, Bouldin et al. (2006)reported decreased root growth of Juncus effusus plants ex-posed to atrazine in a hydroponic solution despite any indica-tions of observable stress. However, Lytle and Lytle (1998)found that Spartina alterniflorawas highly tolerant to atrazinedoses as high as 3.1 mg L−1, whereas the growth of Juncusroemerianus was significantly inhibited at 3.8 mg L−1 (Lytleand Lytle 2005). The results of these and nutrient enrichmentstudies (Valiela et al. 1976; Darby and Turner 2008a, b;Bodker et al. 2015) indicate that concerns about the effectsof atrazine and nutrient loads on the health of the belowgroundbiomass of wetland plants are warranted. To our knowledge,the interactive effects of nutrient loading and atrazine expo-sure on the biomechanical properties of the belowground bio-mass of wetland plants have not been explored.

Wetland macrophytes are subjected to uprooting forcesexerted by wind, waves, gravity, buoyancy, and herbivoregrazing and these forces are resisted by tensile root strength.Tensile strength is the resistance of material in tension to anexternal load (Niklas 1992; Niklas and Spatz 2012). Thestrength of individual roots may be affected by intrinsic fac-tors such as tissue composition, cell wall construction,species-specific anatomical attributes, root turgor pressure,osmotic potential, and plant adaptations to environmental con-ditions (Niklas 1992; Niklas and Spatz 2012). Therefore,chemical compounds exerting anatomical, physiological, ormetabolic changes in plants may have the potential to affectthe tensile strength of plant structures. One way to investigatethese effects is to measure how much tensile root strength isaltered with exposure to increased nutrients and atrazine.

Here we examine the tensile root strength of Spartinapatens (Ait.) Muhl., − a dominant emergent macrophyte ofcoastal wetland plant communities in the Atlantic and Gulfcoasts of the United States. S. patens occupies 96% ofLouisiana’s brackish and intermediate marshes (Chabreck1972), and these two marsh types comprise 54% ofLouisiana coastal marshes (Sasser et al. 2014). Therefore, un-derstanding the effects of xenobiotics on the biomechanicalproperties of S. patens is important for addressing coastal ero-sion. This species is exposed to both atrazine and nutrientloads from agricultural activities in the Mississippi River wa-tershed and from local sources in the Mississippi River Delta(Welch et al. 2014; USEPA 2016). Atrazine exposure andnutrient addition may have synergistic effects onS. patens by atrazine inhibition of the production of ATPand the possible reduction or cessation of root growth andproduction due to excess nutrient addition. Therefore, weexpect these synergistic effects to reduce the tensile rootstrength S. patens. The objective of this study was toinvestigate the effects of interactions between six levelsof nutrients and three levels of atrazine on the tensile rootstrength of S. patens. We conducted greenhouse experi-ments that tested the hypothesis that atrazine and nutrientaddition have synergistic effects on the belowground bio-mass of S. patens.

Materials and Methods

Atrazine-Nutrient Interaction Experiment

Plants were grown under natural light in Louisiana StateUniversity (LSU) greenhouses at Baton Rouge, Louisiana.The experimental design consisted of a 6x3x4 factorial designwith nutrient level and atrazine treatment as the main effects.There were six levels of nutrient addition, three levels of atra-zine exposure, and four replicates of each experimental unit.Spartina patens plugs from Tampa Bay estuary were pur-chased from Green Seasons Nursery (Tampa, Florida, USA).Each plug consisted of 7 to 12 stems growing from a 3.0 ×3.0 × 6.6 cm root mass. These plants did not have a pre-experiment exposure to atrazine. We created a soil mixturefrom commercially available peat moss and natural soil tomimic the soil texture of a coastal wetland soil. The plantsamples were transplanted to 3.78 L (1 gal) glass jars filledwith 3.0 L of a mixture of 65% sphagnum peat (PremierSphagnum Peat Moss; 100% Canadian peat moss, no addedfertilizer or nutrients), 30% clay/silt mixture, and 5% sand.The sand, silt, and clay components were obtained by LSUgreenhouse staff from soil in the Sterlington soil series(coarse-silty, mixed thermic Typic Hapludalfs) located in theMississippi River floodplain in West Baton Rouge Parish,Louisiana. The soil texture of clay/silt components was

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estimated by the texture-by-feel field technique and deter-mined to be sandy clay loam.

The nitrogen and phosphorus nutrient treatments consistedof granular reagent grade calcium nitrate tetrahydrate[Ca(NO3)2 • 4H2O] and granular laboratory grade potassiumphosphate [K3PO4] (Fisher Scient if ic; Nazareth,Pennsylvania). Nutrient treatments were added bi-monthly ina 1 L deionized water solution: High Nitrogen (HN,5.0 mg L−1), Low Nitrogen (LN, 1.75 mg L−1), HighPhosphorus (HP, 0.30 mg L−1), Low Phosphorus (LP,0.10 mg L−1), High Nitrogen x Low Phosphorus (Np), andLow Nitrogen x High Phosphorus (nP). The atrazine treat-ments added bi-monthly in a 1 L deionized water solutionwere: High (3.0 micrograms per liter [μg L−1]), Medium(1.5 μg L−1), and Low (0.5 μg L−1). In addition to untreatedeight control replicates with soil and plants, there were fourdeionized water disturbed ‘controls’ that consisted of a 1 Ldeionized water-atrazine solution into which 1.5 μg L−1 atra-zine treatments were added bi-monthly to ascertain the effectsof sunlight on atrazine molecules. The transplants were accli-mated for 8 weeks to adjust to greenhouse conditions. Glasspots were rotated monthly during the experiment on a reverse-orientation basis (e.g., south to north, west to east) to reducethe variation in environmental conditions. The water levelsbetween treatments were maintained 1.75 cm above the soilsurface to ensure saturated soil conditions. Soil temperature,pH, and redox potential were measured monthly before theaddition of the second bi-monthly nutrient and atrazine treat-ments. We measured each parameter at a depth of 10 cm. Soiltemperature was measured by inserting a soil probe thermom-eter into each unit and recording the result to the nearest 0.1°Celsius (C). The pH of the soil pore water was obtained bywithdrawing a 175 mL sample of soil pore water with a Lislevacuum pump (Lisle Corporation, Clarinda, Iowa) and dis-pensed into a 250 mL amber glass bottle, which was measuredby a Hach HQ 40d multi-parameter meter (Hach IndustriesLoveland, Colorado). The redox potential was measured with45 cm-long standard platinum probes following the proce-dures of Reddy and Delaune (2008) and a Corning calomelreference probe (Corning, Inc. Corning, NewYork) connectedto a Fluke 73Multimeter (John Fluke Manufacturing, Everett,Washington). A correction of +244 mV was added to redoxmeasurements (Reddy and Delaune 2008). The experimentwas conducted for a total of 212 days from 1 December2015 until 30 June 2016.

Disturbed Controls Experiment

We conducted a disturbed control experiment to monitor theimpact of the atrazine and nutrient addition main effects on theplant samples. The experimental design consisted of eightreplicates of each of the six nutrient and three atrazine treat-ments that were used for the atrazine-nutrient interaction

experiment, plus 8 control replicates for each main effect.The plant samples, soil components, environmental con-ditions, and experimental set-up were the same as theatrazine-nutrient interaction experiment, except that theatrazine treatments were added weekly. The experimentwas conducted for a total of 60 days from 1 December2015 until 30 January 2016.

Tensile Strength Testing

We tested the tensile strength of live roots in only one ofthe five diameter size classes utilized by Hollis and Turner(2018). The Small size class (0.5–1.0 mm) was selectedbecause of the relatively high numbers of roots within thisdiameter range and the increased probabili ty ofconducting successful tensile strength tests. A mean ofsix tests were conducted for every successful tensilestrength test. A successful test consisted of root samplesthat failed between the supports of the test stand, whereasroots that failed at the supports were considered an invalidtest. Live roots and rhizomes were differentiated fromdead roots by their white, turgid, and translucent appear-ance, whereas dead roots are dark and flaccid (Darby andTurner 2008a). However, many live roots were stained bysoil deposits and they were separated from dead roots bythe presence of turgor, bifurcations of fine roots, and theirability to float.

We measured three individual root metrics: mass, length,and diameter; the cross-sectional area and volume were calcu-lated from these metrics. We measured root length to thenearest 0.1 mm with a Scale Master© Classic digital planim-eter (Calculated Industries, Carson, Nevada USA). We mea-sured the mean root diameter to the nearest 0.1 mm with aStarrett digital IP67 micrometer. Measurements were taken atboth ends and at the middle of each root and then averaged.We calculated the cross-sectional area (mm2) and volume(mm3) from the length and diameter measurements after ten-sile strength testing was performed. Root samples wereweighed to the nearest 0.1 mg. We used a MecmesinMultiTest 1–d motorized stand (Mecmesin Limited; Sinfold,West Sussex, United Kingdom) to measure tensile rootstrength (as breaking force) in Newtons (N). Individual rootswere secured to two support clamps aligned perpendicular tothe base of the test stand. The contact surfaces of the clampsprovided 1.25 × 2.50 cm of area and were lined with finesandpaper to reduce or eliminate slippage. In addition, thesupport clamps were attached to a Mecmesin Basic ForceGage load meter, which was capable of measuring 1000 Nof force with a precision of 0.1 N. The test stand was activatedand the top support was pulled upward by a vertical hydraulicpiston until the root exhibited structural failure. The load thatinduced failure at that point, or breaking force, was recordedas the tensile root strength.

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Tissue Sample Testing

Samples of live stem, leaf, and root tissue were collected aftertensile strength testing at the end of the experiments and sentto the LSU Soil Testing and Plant Analysis Laboratory fordetermination of the carbon, nitrogen, and phosphorus tissuecontent. The stem and leaf tissue of each experimental unitwere combined as an aboveground biomass sample for thatunit and the roots comprised the belowground biomass sam-ple. Tissue samples were separated by treatment, dried at65 °C for 48 h, and homogenized. These results were usedto calculate carbon-nitrogen (C:N) and nitrogen-phosphorus(N:P) ratios. The LSU Department of Agricultural Chemistryanalyzed water, soil, and root samples for atrazine,deethylatrazine, and deisopropylatrazine concentration withgas chromatography-mass spectrometry (GC-MS) analysisusing standard operating procedures that were modified fromUnited States Environmental Protection Agency (USEPA)Methods 525, 507–1, and 507–2.

Statistical Analyses

We conducted a one-way analysis of variance (ANOVA)(JMP v. 13, SAS Institute, Cary, North Carolina) to test forsignificant differences between the nutrient and atrazine maineffects and their respective controls in the disturbed controlsexperiment. We also used a Tukey-Kramer Honest SignificantDifference (HSD) test in both experiments, to determine ifthere were any significant differences between the tensile rootstrengthmeans. The data are reported as the mean ± 1 standarderror of the mean unless otherwise noted. Homoscedasticityand normality of residuals were determined with Brown-Forsythe and Shapiro-Wilks tests, respectively. The data thatdid not meet the assumptions of an ANOVAwere tested with aWelch’s ANOVA, and the differences between the tensilestrength means were determined using a Steel-Dwass non-parametric multiple comparison test.

We used a Welch’s ANOVA in JMP v. 13 to test for differ-ences in the mean tensile strength of roots by nutrient additionand atrazine treatment in the atrazine-nutrient interaction ex-periment. We could not use a two-way ANOVA to test forinteractive effects because the data violated assumptions ofboth normality and homoscedascity. Therefore, we tested forinteractive effects by segregating the tensile root strength dataof the levels of one main effect into subsets, and then conduct-ed a one-way Welch’s ANOVA of tensile root strength usingeach level of the other main effect. For instance, the tensileroot strength data were divided by the three levels of the atra-zine main effect into High (3.0 μg L−1), Medium (1.5 μg L−1),and Low (0.5 μg L−1) subsets and then one-way ANOVAs oftensile root strength were conducted for each of the six levelsof the nutrient addition main effect (e.g., Tensile strength xHigh Nitrogen using the High atrazine data subset). In

addition, the presence of any interactive effects of treatmentcombinations was also determined by using a Kolmogorov-Smirnov goodness-of-fit test to compare the data distributionof the nutrient-atrazine combination treatment with that of thestrongest main effect of the treatment combination (e.g. Maineffect A vs. combination treatment AxB).

A Student’s t test was used to test for the existence of astatistical significance among the soil temperature, redox po-tential, and pH parameters. The differences among the nutrientand the Carbon: Nitrogen: Phosphorus (CNP) ratios were test-ed with a one-way ANOVA. All statistical tests were per-formed at a significance level of p < 0.05.

Results

Disturbed Controls Experiment

A one-way Welch’s ANOVA detected no significant dif-ference in tensile root strength in either the atrazine treat-ments or Control (Fig. 1a, F = 1.002, p = 0.393) or in thenutrient treatments and Control (Fig. 2a, F = 1.076, p =0.381) after 60 days. In addition, there was no significantdifference in tensile root strength among the atrazine ornutrient treatments. The means of the tensile root strengthof the atrazine and nutrient treatments were 4.6 ± 0.30 Nand 4.4 ± 0.39 N, respectively.

Atrazine-Nutrient Interaction Experiment

Soil Parameters

The mean soil temperature in the experimental units rangedfrom 26.1 to 26.6 °C (Appendix 1, Fig. 7; Appendix 2 Table 3)with an overall mean of 26.3 ± 0.41 °C, and less than 1 °Cvariation between the mean temperatures for each soil texture.A Student’s t test revealed no significant difference betweenthe soil temperatures among the High (t = 2.2, df = 12; P =0.97), Medium (t = 2.2, df = 12; P = 0.98), and Low (t = 2.2,df = 12; P = 0.99) atrazine treatments or Control.

The pH of the experimental units was neutral to alkalinethroughout the experiment and the mean pH was 7.1 in allthree atrazine treatments (Appendix 1, Fig. 8; Appendix 2Table 3). As a result, a Student’s t test found no significantdifferences between the High (t = 2.3, df = 8; P = 1.0),Medium (t = 2.3, df = 8; P= 0.78), and Low (t = 2.2, df = 10;P= 1.0) atrazine treatments or Control.

The redox potential fluctuated frequently throughout theduration of the experiment, but there was less than 6 mV ofvariation between the redox potential means of the experimen-tal units and control (Appendix 1, Fig. 9; Appendix 2 Table 3).Consequently, a Student’s t test revealed no significant differ-ences in the soil redox potential among the High (t = 2.2, df =

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10; P = 0.52), Medium (t = 2.2, df = 10; P = 0.48), and Low(t = 2.2, df = 12; P = 0.32) atrazine treatments and Control.

Tensile Root Strength

A one-way Welch’s ANOVA detected significant differences inthe tensile root strength between all atrazine treatments andControl (Fig. 1b, F = 18.9, p < 0.0001) after 212 days; however,there were no significant differences among the tensile rootstrength of the atrazine treatments, and the grand tensile root

strength mean was 2.07 ± 0.30 N, compared to 4.19. ± 0.21 Nin the Control units. A one-way Welch’s ANOVA revealed asignificant difference in the tensile root strength between all nu-trient treatments and Control (Fig. 2b, F = 12.6, p < 0.0001) after212 days, as well as significant differences between the tensileroot strength of the LP andHP treatments (p = 0.032) and the Npand HP (p = 0.037) treatments (Appendix 2, Table 1, Table 2).

A one-way Welch’s ANOVA of the High Nitrogen (HN)and Low Nitrogen (LN) subsets revealed significant differ-ences in tensile root strength between all atrazine treatmentsand Control (Fig. 3a, F = 16.3, p < 0.0001; Fig. 3b, F = 23.1,p < 0.0001, respectively); however, there were no significantdifferences among the tensile root strength of the atrazinetreatments for either subset. The grand means of tensile rootstrength for the HN and LN subsets were 2.60 ± 0.22 and 2.36

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Fig. 1 Box and whisker plots of (a) a one-way ANOVA of tensile rootstrength with atrazine as the main effect for the 60-day atrazine controlgreenhouse experiment. There was no significant difference between con-trol and atrazine treatments or among atrazine treatments (p = 0.393) (b)one-way Welch’s ANOVA of tensile root strength with atrazine as themain effect for the 212-day atrazine-nutrient interaction greenhouse ex-periment. Tensile root strength in the control (0 μg L−1) was significantlyhigher than in low (0.5 μg L−1), medium (1.5 μg L−1), and high(3.0 μg L−1) atrazine treatments (Appendix Table 1, F = 18.9,p < 0.0001). There were no significant differences between the atrazinetreatments (p = 0.39). The box plot whiskers represent the sample range;the blue horizontal lines denote ±1 standard deviation; the center horizon-tal red lines represent the group mean ± 1 standard error of the mean. Thehorizontal green line is the grand mean for all groups. Box plots withdifferent letters denote significant differences between treatments

60 days

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Fig. 2 Box and whisker plots of (a) a one-way ANOVA of tensile rootstrength with nutrient addition as the main effect for the 60-day nutrientcontrol greenhouse experiment. There was no significant difference be-tween control and nutrient treatments (p = 0.381) or among nutrient treat-ments (p > 0.05) (b) one-way Welch’s ANOVA of tensile root strengthwith nutrient addition as the main effect for the 212-day atrazine-nutrientinteraction greenhouse experiment. The tensile root strength in the controlwas significantly higher than in the nutrient treatments (F = 12.6,p < 0.0001). There were no significant differences between the atrazinetreatments (p > 0.05). The box plot whiskers represent the range; the bluehorizontal lines denote ±1 standard deviation; the center horizontal redlines represent the group mean ± 1 standard error of the mean. The hori-zontal green line is the grand mean for all groups. Box plots with differentletters denote significant differences between treatments

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± 0.21 N, respectively, which was a 38 to 44% loss in tensileroot strength compared to the Control.

A one-way Welch’s ANOVA of the High Phosphorus (HP)and Low Phosphorus (LP) subsets revealed significant differ-ences in tensile root strength between all atrazine treatmentsand Control (Fig. 3c, F = 27.0, p < 0.0001; Fig. 3d, F = 22.2,p < 0.0002, respectively). There were significant differences be-tween the tensile root strength of the High andMedium atrazinetreatments for the HP subset (p = 0.049), as well as between theHigh and Low atrazine treatments for the LP subset (p = 0.003).The tensile root strength grand means for the HP and LP subsetswere 2.31 ± 0.22 and 2.70 ± 0.23 N, respectively, which was a36 to 45% loss in tensile root strength compared to the Control.

Significant differences in tensile root strength between allatrazine treatments and Control were revealed by a one-wayWelch’s ANOVA of tensile root strength in the nitrogen-phosphorus combination subsets (Np and nP) (Fig. 3e, F =14.2, p < 0.0001; Fig. 3f, F = 20.8, p < 0.0001, respectively);

however, there were no significant differences among the ten-sile root strength atrazine treatments for either nutrient subset(p > 0.05). The tensile root strength grand means for the Npand nP subsets were 2.69 ± 0.27 and 2.40 ± 0.21 N, respec-tively, which was a 36 to 43% loss in tensile root strengthcompared to the Control.

A one-wayWelch’s ANOVAof the High atrazine treatmentsubset found significant differences in tensile root strengthbetween all nutrient treatments and Control (Fig. 4a, F =15.9, p < 0.0001); there were significant differences in tensileroot strength between the LP and the LN, HP, nP, and Npnutrient treatments (p < 0.03). The tensile root strength grandmean for the High subset was 2.28 ± 0.21 N, or a 46% loss intensile root strength.

In the Medium atrazine subset there were significant differ-ences in tensile root strength between all nutrient treatmentsand Control (Fig. 4b, F = 16.4, p < 0.0001); but there were nosignificant differences in tensile root strength among the

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Fig. 3 Box and whisker plots ofone-way Welch’s ANOVA oftensile root strength with atrazineas the main effect for (a) the HighNitrogen (HN) (b) the LowNitrogen (LN) (c) HighPhosphorus (HP) (d) LowPhosphorus (LP) (e) HighNitrogen-Low Phosphorus (Np)(f) Low Nitrogen-HighPhosphorus (nP) treatment sub-sets to test for interactive effectsbetween nutrient and atrazinetreatments. There were significantdifferences between control andatrazine treatments for all subsets(Appendix Table 1, p < 0.0001).The box plot whiskers representthe range; the blue horizontallines denote ±1 standard devia-tion; the center horizontal redlines represent the groupmean ± 1standard error of the mean. Thehorizontal green line is the grandmean for all groups. Box plotswith different letters denote sig-nificant differences betweentreatments

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nutrient treatments (p > 0.05). The tensile root strength grandmean for the Medium subset was 2.35 ± 0.21 N, or 44% lowerthan in the Control.

A one-way Welch’s ANOVA of the tensile root strength inthe Low atrazine treatment subset found significant differ-ences between all nutrient treatments and Control (Fig. 4c, F= 17.9, p < 0.0001). In addition, there were significant differ-ences in tensile root strength among the Np and the LN (p =

0.004) and HP (p = 0.042) nutrient treatments. The tensile rootstrength grand mean for the Low subset was 2.18 ± 0.21 N, or48% lower than in the Control.

Nutrient Tissue Content

The carbon content of S. patens above- and belowground tissuevaried between the nutrient treatments. With the exception ofthe High Nitrogen (HN) treatment, a greater concentration ofcarbon was detected in the aboveground (stem) tissue than inthe roots (Appendix 2, Table 4). A one-way ANOVA revealedthat the carbon content in the stems for the High and LowPhosphorus (HP and LP) and Low Nitrogen-HighPhosphorus (nP) units were significantly higher than theControl (F = 12.9, p < 0.0001), but that the carbon content ofplants in the LN and HN units were not significantly differentthan in the Control (p > 0.05). The C:N ratio in the roots wasless than 100; however, the C:N ratio in the stems ranged from85 in the nP units to 100.9 in LN units. Similarly, there weregreater concentrations of nitrogen and phosphorus in the rootsthan in the stems. The N:P ratios in the roots ranged from 7.2 inthe HP units to 10.6 in the Control. The N:P ratios in the stemsranged from 9.6 in the HN and LN units, respectively, to 11.3 inthe Low Nitrogen-High Phosphorus (nP). A one-way ANOVArevealed that the nitrogen content in the roots for the LP and nPunits was significantly higher than in the Control (F = 7.9,p < 0.0001). However, with the exception of the HN treatment,the N:P ratios in the stems were significantly higher than in theroots (F = 7.5, p = 0.018).

Atrazine Levels

Neither atrazine nor any of its primarymetabolites were detectedin leaf, root, or solid soil samples from any of the Low,Medium,or High atrazine experimental units. The detection limit for plantbiomass and soil samples was 25μg L−1; however, the detectionlimit for soil porewater samples was <0.1 μg L−1. Atrazine wasdetected in the soil porewater of the Low, Medium, and Highatrazine units at a concentration of 0.0083 μg L−1,0.0095 μg L−1, and 0.0435 μg L−1, respectively. In addition,atrazine and DEAwere detected in the deionized water controlsat mean concentrations of 6.96 and 1.60 μg L−1, respectively.

Discussion

Atrazine Exposure

Atrazine was detected in the soil porewater at concentrationsless than 0.1 μg L−1 in all three treatment units. In addition,atrazine and deethylatrazine (DEA) were detected in the deion-ized water controls at concentrations above the range of thetreatment levels (1.60 to 6.96 μg L−1 vs. 1.0 to 5.0 μg L−1).

Fig. 4 Box and whisker plots of one-wayWelch’s ANOVA of tensile rootstrength with nutrient addition as the main effect for (a) the High Atrazine(b) the Medium Atrazine (c) Low Atrazine treatment subsets to test forinteractive effects between nutrient and atrazine treatments. There weresignificant differences between control and atrazine treatments for allsubsets (Appendix Table 1, p < 0.0001). The box plot whiskers representthe range; the blue horizontal lines denote ±1 standard deviation; thecenter horizontal red lines represent the group mean ± 1 standard errorof the mean. The horizontal green line is the grand mean for all groups.Box plots with different letters denote significant differences betweentreatments

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However, the herbicide was not detected in the leaf, root, or soilsamples (25 μg L−1 detection limit). These results suggest thatatrazine did not undergo rapid photodegradation in the watercolumn, and the lack of detection in the soil samples indicatesthat adsorption may not have been a major contributor to thefate of atrazine doses. On the other hand, the soil porewaterresults may be an indication of desorption of atrazinemoleculesthat had been adsorbed by the organic-dominated soil.Furthermore, the absence of the primary metabolites in thesoil and water column samples suggests that the atrazinedoses were not present in these areas long enough to undergotransformation. Davis et al. (1965) reported that the uptake ofatrazine in corn occurred in period of 12 to 100 h, which sug-gests that given the duration of this experiment, it is highlylikely that the herbicide was assimilated by S. patens.

The solubility of atrazine can be affected by temperature. Astemperatures increase, atrazine becomes more soluble becauseof conditions that are conducive to severing molecular bonds(McGlamery and Slife 1966) and its availability to plants in-creases. Therefore, the soil temperature in the experimental unitsmay have facilitated atrazine uptake and a subsequent decline intensile root strength. Atrazine adsorption and degradation aregenerally curtailed under anaerobic conditions, but they are rap-id under aerobic conditions (McGlamery and Slife 1966).

An oxidized rhizosphere may have provided pockets alongthe root channels where atrazine assimilation, degradation,and/or transformation were possible. Consequently, radial ox-ygen loss (ROL) from the roots could have nullified the effectof the anaerobic conditions on atrazine adsorption, whichwould have allowed plant uptake and subsequent effects ontensile root strength.

The pH of the soil may have affected tensile root strengthbecause of its influence on atrazine availability. The adsorptionof atrazine onto organic and mineral soil colloids may be affect-ed by pH. Ionized humic acids can adsorb protonated atrazinemolecules by ionic bonding (Senesi 1992), whereas less atrazineadsorption occurs under alkaline conditions (McGlamery andSlife 1966). The mean pH in all experimental units was main-tained at or above 7.0 pH for the duration of the experiment. Asa result, there may have been a higher probability of atrazineavailability and lower rates of adsorption, if any adsorption oc-curred at all. However, the availability of adsorbed atrazine is afunction of time and pH; the longer the herbicide molecules arebound to the substratum, then the more time and energy will berequired to extract them (Mandelbaum et al. 2008).

Nutrient Addition

Nutrient cycling can be influenced by the effects of soil texture,soil temperature, pH and redox potential. For instance, soil tex-ture is a major driver of soil saturation and field capacity condi-tions (bothmicropores andmacropores are flooded)may change

the biogeochemistry of the soil. Anaerobiosis affects the fate ofnitrogen species such as nitrate, which may be reduced by de-nitrification. Soil temperature can affect the rates of chemicalreactions, which can double with every 10 °C increase in tem-perature. Consequently, the increased respiration rates and nutri-ent cycling can exact a carbon demand on the plant. The deni-trification of nitrate in the experimental units would have re-quired carbon as an electron donor and the tensile root strengthmay have been affected by the loss of structural material as theadditional electron acceptor added to the experimental units(e.g., nitrate in calcium nitrate tetrahydrate) provided the catalystfor direct or indirect use of plant tissue as a carbon donor and,ultimately, decreased root tensile root strength.

The soil pH may alter nutrient dynamics as well. For in-stance, acidic conditions can facilitate the precipitation of phos-phorus frommetal complexes with iron. The pH also affects thepartitioning of a compound between the solute and solution(McGlamery and Slife 1966). The soil pH can affect nutrientcycling indirectly by directly affecting microbial communities,which are also sensitive to the redox potential of the soil(Fenchel et al. 2012). The balance between nitrogen and phos-phorus uptake may have also affected tensile root strength. Themolar N:P ratios for all experimental units were < 12, which isan indication that nitrogen was limiting aboveground growth.However, as shown in Appendix Table 4, the addition of phos-phorus, even at the lowest dose (LP) resulted in an increase inthe nitrogen concentration of roots. Furthermore, resourcepartitioning of nutrients between the above- and belowgroundbiomass indicates that the bulk of the assimilated nitrogen wasstored as robust aboveground biomass, while the phosphorusconcentrated in the atrophied belowground biomass. Also,phosphorus may accumulate in the plant tissue because thereis no biogeochemical process such as denitrification to removeit from the system. The surfeit of nutrients may have curtailedthe growth of the roots in the experimental units, which isconsistent with the Marginal Value Theorem of the OptimumForaging Theory (McNickle and Cahill Jr 2009).

Nutrient-Atrazine Combination Treatments

The combination of nutrient addition and atrazine exposuredrastically altered the root architecture of the treated plants(Fig. 5), and the effect was the same, no matter the combination(e.g., High atrazine x Low Phosphorus or Low atrazine x Highnitrogen). During the 60-day atrazine experiment, atrazine wasadded to S. patens samples on a weekly basis for 8 weeks. Themean tensile root strength for the units in this experiment was4.41 ± 0.43 N, whereas the mean tensile root strength of thenutrient treatment units in the High atrazine subset in the inter-action experiment was 1.96 ± 0.20 N. It is important to note thatthe main difference between the two experiments was only theapplication of atrazine; the soil texture and hydrologic regimes

766 Wetlands (2019) 39:759–775

were virtually identical. The differences in soil parameters werenot statistically significant. The soil temperature varied by1.5 °C (due to seasonal variations, despite greenhouse controls),the soil pH varied by 0.1 units, and the soil redox potentialvaried by 14.2 mV. In the first atrazine experiment, a total of24 μg L−1 were added to the plants (High dose) over eightweeks; whereas in the nutrient-atrazine interaction experiment,a total of 42 μg L−1 of atrazine were added in 28 weeks.However, the frequency of the added doses did not seem to bethe difference in the outcome of the two experiments. The per-sistence of the herbicide in the rhizosphere and inside the plantmay be one of the key factors that caused reduced tensile rootstrength. As a result, it seems that 1) there is a temporal compo-nent to the effects of atrazine, and 2) the impact on S. patensdoes not occur immediately, even though the uptake of atrazinemay occur rapidly. Atrazine may be sorbed and desorbed to soilparticles and the rate of adsorption and desorption may vary,which is an indication of hysteresis and a lack of equilibriumbetween the herbicide and the soil and water fractions (Clay andKoskinen 1990a). However, the addition of nutrients, especiallyphosphorus, seemed to exacerbate the effects of atrazine expo-sure on the plants. The atrazine-HP and atrazine-nP units pro-duced the lowest group mean tensile root strengths of the entireexperiment (Appendix Table 1, 1.69 ± 0.22 N and 1.76 ± 0.21 Nvs. Control at 4.19 ± 0.21 N) and the HP level alone producedthe lowest mean tensile strength for an individual treatment(1.32 ± 0.20 N). The effects of the nitrogen-phosphorus combi-nation in concert with atrazine exposure are demonstrated mostemphatically by results shown in Fig. 5a, b. The experimentalunits clearly lack the biomass of the control, and rhizome devel-opment was nonexistent. The root biomass may have atrophiedbecause of carbon loss due to respiration as well as curtailedgrowth due to surplus nutrients and photosynthesis inhibitionby atrazine exposure. The consequences of carbon demandand lack of replenishment of root biomass may be manifestedby reduced tensile strength. In addition, the lack of rhizomeproduction would have severe biomechanical consequencesfor the plant and the wetland ecosystem. The plants inFig. 6a–d could be easily uprooted from the soil because offewer rhizomes and lower fine root production. The lack ofrhizomes on the experimental units may indicate reduced fitness

�Fig. 5 The belowground biomass production for the Control vs. the (a)Medium Atrazine x Low Nitrogen experimental unit, and (b) MediumAtrazine x Low Phosphorus experimental unit in the 212-day atrazine-nutrient interaction experiment. The tensile root strength of the M x LN(2.02 ± 0.23, p < 0.0001) was significantly weaker than Control (4.19 ±0.23); (b) Medium Atrazine x Low Phosphorus experimental unit in the212-day atrazine-nutrient interaction experiment. The tensile rootstrength of the M x LP (2.14 ± 0.23, p < 0.0001) was significantly weakerthan Control (4.19 ± 0.23). Note the lack of rhizomes and decreased num-ber of fine roots on both experimental units. The polygons with the whitedotted lines delineate the root biomass present at the beginning of theexperiment

Wetlands (2019) 39:759–775 767

because of the inability of the plant to store photosynthate orproduce new ramets. In addition, the lack of rhizomes wouldseverely weaken the biomechanical stability of the plant and soildue to inability to generate new lateral roots with subsequentfine roots and roots hairs (Niklas 1992; Niklas and Spatz 2012).As a result, soil-plant friction could be greatly reduced, whichalso decreases the volume of soil that is reinforced by roots andleads to weaker soil shear strength (Turner 2011). The reducednumber of roots could result in the additional loading of tension-al forces on fewer roots with much less soil-plant friction, whichcould make them more susceptible to failure (Niklas 1992;Niklas and Spatz 2012). Furthermore, the magnitude of forcesneed to uproot the plant may be considerably less with the ab-sence of rhizomes and the reduced root architecture.

Interactive Effects

The interactive effects between two substances may be de-fined as the presence of main effect A affects the activity ofmain effect B. If the effects of the combination of A and B

(AxB) are greater than that of the greater of either A or Balone; then there are interactive effects of A and B. TheKolmogorov-Smirnov goodness-of-fit test tested the null hy-pothesis that the distributions of the atrazine-nutrient treat-ment combination and main effects tensile root strength datawere not different. However, interactive effects were detectedin two out of 18 Kolmogorov-Smirnov tests, which suggestthat it is unlikely that there were interactive effects betweenthe six nutrient levels and three atrazine doses.

Ecological Implications

Emergent macrophytes may function as foundation species inwetland and aquatic ecosystems. They are primary producersthat form the foundation of food webs by providing forage forinvertebrate and vertebrate species. In addition, emergent mac-rophytes are a source of organic carbon for bacteria and theirabove- and belowground biomass can serve as a substrate forperiphyton. They provide the structural stability that allowscoastal wetlands to occupy a position between marine and

Fig. 6 Additional examples of the belowground biomass production forthe Control vs. the (a) High Atrazine x Low Nitrogen (H x LN) (b) HighAtrazine x Low Phosphorus (H x LP) (c) Low Atrazine x LowPhosphorus (L x LP) (d). Medium Atrazine x Low Nitrogen-HighPhosphorus (M x nP) experimental unit in the 212-day atrazine-nutrientinteraction experiment. The tensile root strength of both the L x LP (1.66

± 0.21, p < 0.0001) and M x nP (1.54 ± 0.23, p < 0.0001) units were sig-nificantly weaker than Control (4.19 ± 0.23). Note the lack of rhizomesand decreased number of fine roots on the experimental units. The poly-gons with the white dotted lines delineate the root biomass present at thebeginning of the experiment

768 Wetlands (2019) 39:759–775

terrestrial habitats. Emergent macrophytes can influence wet-land development by altering hydrology, trapping suspendedsediment, and providing habitat for ecosystem engineers suchas alligators and beaver. Coastal wetlands serve as vital nurseryhabitat for marine and estuarine fishes and invertebrates, manyof which are commercially valuable species such as brown(Farfantepenaeus aztecus) and white (Litopenaeus setiferus)shrimp, blue crab (Callinectes sapidus), oysters (Crassostreavirginica), redfish (Sciaenops ocellatus), speckled trout(Cynoscion nebulosus), and flounder (Paralichthyslethostigma). In addition, coastal wetlands provide winteringand stopover habitat for numerous species of waterfowl,Neotropical songbirds, wading birds, and shorebirds.Therefore, the erosion of coastal wetlands can have profoundecological consequences such as the disruption of trophic dy-namics and biogeochemical cycling and functions. The degra-dation of tensile root strength in wetland emergent macrophytescould place the entire ecosystem in jeopardy. The belowgroundbiomass of emergent species such as S. patens andS. alterniflora provide structural reinforcement of wetland soils,many of which are dominated by organic material. Wetlandsthus serve as a source, sink, or transformer of xenobiotics. Theinfluxes of xenobiotics such as herbicides and nutrients providea massive supply of alternate electron acceptors to a vast reser-voir of carbon that can be used as an electron donor. The deg-radation of belowground biomass and/or tensile root strengthincreases the vulnerability of wetlands to major natural distur-bances such as tropical storms and hurricanes. Without emer-gent vegetation, the accumulated peat in wetland soils maycollapse and expose coastal wetlands to inundation by the sea.The loss of coastal wetlands would mean the loss of the impor-tant ecological functions that they perform, such as the filtering,sequestration, and transformation of chemical compounds.Consequently, xenobiotics may disrupt ecosystem processesfurther by affecting the phytoplankton, which are another majorsource of primary production. Eutrophication may facilitate ashift in phytoplankton communities as marine species displaceor outcompete estuarine and fresh water species. The increasein nitrogen and phosphorus in coastal waters, accompanied by adecline in silica, can shift N:S and P:S ratios and alter thecomposition of phytoplankton communities (Howarth et al.2000; Turner et al. 2008). The disintegration of coastal wetlandswould remove a significant means of improving the quality ofwater that flows to coastal areas, which may create a positivefeedback loop that could increase the frequency and distributionof harmful algal blooms. Furthermore, eutrophic nutrient levelsin freshwater inflows have been shown to increase the size andpersistence of hypoxic or anoxic ‘dead zones’ in estuarine andnearshore areas (Rabalais 2009). These low oxygen areas cankill or displace benthic, demersal, and pelagic species and altertrophic dynamics. Eutrophication can also increase water tur-bidity by stimulating the growth of epiphytes and macroalgae,which would inhibit light penetration into the water column

(Zieman and Zieman 1989). As a result, the lower light levelsmay degrade seagrass beds and exacerbate ecological damagein estuarine and nearshore areas. The loss or degradation ofseagrass beds would reduce the amount of reproduction, nurs-ery, and foraging habitat that seagrasses provide for benthic,demersal, and pelagic communities, which could adversely af-fect the economic status of human coastal communities.

The loss of coastal wetlands could result in the completecollapse of stocks of commercially valuable marine and estu-arine species because of the synergistic effects of eutrophica-tion due to the loss of estuarine and nearshore nursery habitatand toxic nearshore habitat for adults. Therefore, the biophys-ical status of coastal emergent macrophytes is of the utmostimportance for coastal and marine ecosystems as well as hu-man communities that are reliant on these resources for eco-nomic activities and personal well-being. The biomass of wet-land plants are a natural defense against large natural distur-bances such as tropical cyclones. However, wetlands that arecomprised of vegetation with weak tensile root strength maybe fragmented by storm surge. The loss of the important eco-logical service of wave attenuation could magnify the stormdamage to coastal human communities, as in the case withNew Orleans and the Breton Sound estuary duringHurricane Katrina. Consequently, the implications of thisstudy are that urgent action is needed to mitigate the influxof xenobiotics to coastal wetlands and estuaries.

Conclusions

The tensile root strength of S. patens in these experiments de-clined with exposure to atrazine, nutrients, and in combinationwith both. The phosphorus and the nitrogen-phosphorus combi-nation had the greatest effect on tensile root strength compared tothe other treatments. The effects of the nutrient-atrazine combi-nation produced the lowest recorded tensile root strength. Theroot biomass in the experimental units was visibly smaller than inthe Controls either because biomass decomposed faster or wasnot produced as quickly. The application of atrazine and theaddition of nutrients resulted in roots with decreased tensilestrength and structurally compromised belowground biomassbecause of the sparse rhizome and fine root production. Coastalmacrophytes need the biomechanical reinforcement of roots toresist powerful natural disturbances. Even if the plants are notdislodged from the marsh, the loss in belowground biomass willcurtail the wetlands’ ability to accrete new organic matter andkeep pace with relative sea level rise. Atrazine exposure hasecosystem-level implications that are beyond the effects it hason the dominant vegetation. As an herbicide, atrazine may alsoaffect phytoplankton and cause ecological damage at other tro-phic levels, including those of commercially valuable estuarineand marine species. The Louisiana coast receives atrazine fromagricultural fields in the upper Mississippi River watershed that

Wetlands (2019) 39:759–775 769

may affect marine species as well as wetland restoration out-comes within the Holocene floodplain of the Mississippi River.

Acknowledgements We thank Erick Swenson for logistical support andCharles Milan for laboratory assistance. Funding for this research wasprovided by the Louisiana Board of Regents Graduate StudentFellowship and Shell Biodiversity in Wetlands Graduate Student SupportFund. We appreciate discussing the statistical analyses with Ed Laws.

Appendix 1: Soil Parameter Results

Soil Temperature

A Student’s t test revealed no significant difference between thesoil temperatures among the three atrazine treatments or Control(p > 0.05). The mean soil temperature in the experimental unitsranged from 26.1 to 26.6 °C (Fig. 7) with an overall mean of26.3 (± 0.41 °C, SE) and less than 1 °C variation between themean temperature for each soil texture. The highest temperaturewas observed in the Low (31.4 °C) units and the lowest temper-ature was also recorded in the Low units (19.7 °C). Soil temper-ature in the experimental units decreased sharply in December2015–January 2016 and April–May 2016, but remained within1 °C of the mean temperature for most of the experiment.

Soil pH

AStudent’s t test found no significant differences between the soilpH among the three atrazine treatments and the Control(p> 0.05). The pH of the experimental units was neutral to alka-line throughout the experimentwhile the control units were slight-ly acidic for two periods in January and March 2016 (Fig. 8).However, the pH of both the experimental and Control unitsfluctuated considerably above pH 7.0 in April–June 2016. Themean pH was 7.1 in all three atrazine treatments and the control.

Redox Potential

The redox potential fluctuated frequently between the ex-perimental units throughout the duration of the experi-ment. There was less than 6 mV of variation betweenthe redox potential means of the experimental units andControl (Fig. 9). Consequently, a Student’s t test revealedno significant differences in the soil redox potentialamong the three atrazine treatments and control(p > 0.05). The experimental units exhibited a range dif-ferential from 15 to 25 ± 0.7 mV. For example, the Lowatrazine treatment units ranged from a minimum of−26.6 mV to a maximum of −3.6 mV, which is a differ-ence of 23 mV. The redox potential in all units also de-clined 10 to 20 mV during April–May 2016, before in-creasing by 25 mV in June 2016 (Fig. 9). The redox po-tential of the experimental and Control units remained in arange below zero throughout the experiment that was con-ducive to the utilization of iron and manganese as alter-nate electron acceptors.

Fig. 7 The monthly mean soil temperatures for the experimental units inthe 212-day atrazine-nutrient interaction experiment

6.4

6.6

6.8

7.0

7.2

7.4

7.6

7.8

NOV DEC JAN FEB MAR APR MAY JUN

pH

Month

Low

Medium

High

Control

Fig. 8 The monthly mean pH for the experimental units in the 212-dayatrazine-nutrient interaction experiment

-35.0

-30.0

-25.0

-20.0

-15.0

-10.0

-5.0

0.0

NOV DEC JAN FEB MAR APR MAY JUN

Redo

x po

ten�

al (m

V)

Month

Low

Medium

High

Control

Fig. 9 Themonthly mean redox potential for the experimental units in the212-day atrazine-nutrient interaction experiment

770 Wetlands (2019) 39:759–775

Appendix 2: Summary of Tensile RootStrength Results

Table 1 Summary statistics of the tensile root strength response variable for the atrazine treatment and nutrient additionmain effects (in bold), andmaineffect subsets (in bold, subset in parentheses) used to test for interactive effects

Source n Max Min Mean Group Mean Grand Mean SE SD p value

Nutrient 280 n/a n/a n/a 1.95 2.07 n/a n/a < 0.0001

Control 40 9.9 0.8 4.19a n/a n/a 0.21 1.90 n/a

High Nitrogen (HN) 40 6.6 0.2 2.06 bc n/a n/a 0.12 1.22 < 0.0001

Low Nitrogen (LN) 40 5.1 0.3 1.76 bc n/a n/a 0.12 1.05 < 0.0001

High Phosphorus (HP) 40 6.8 0.2 1.69 c n/a n/a 0.12 1.16 < 0.0001

Low Phosphorus (LP) 40 6.8 0.1 2.21 b n/a n/a 0.12 1.34 < 0.0001

High Nitrogen-Low Phosphorus (Np) 40 7.4 0.1 2.20 b n/a n/a 0.12 1.62 < 0.0001

Low Nitrogen-High Phosphorus (nP) 40 6.2 0.2 1.80 bc n/a n/a 0.12 1.03 < 0.0001

Nutrient (Low) 280 n/a n/a n/a 1.85 2.18 n/a n/a < 0.0001

Control 40 9.9 0.8 4.19 a n/a n/a 0.21 1.90 n/a

High Nitrogen (HN) 40 4.8 0.3 1.87 bc n/a n/a 0.21 1.02 < 0.0001

Low Nitrogen (LN) 40 4.7 0.3 1.41 c n/a n/a 0.21 0.89 < 0.0001

High Phosphorus (HP) 40 4.7 0.4 1.64 c n/a n/a 0.21 0.80 < 0.0001

Low Phosphorus (LP) 40 3.2 0.3 1.66 bc n/a n/a 0.21 0.80 < 0.0001

High Nitrogen-Low Phosphorus (Np) 40 7.2 0.3 2.52 b n/a n/a 0.21 1.99 < 0.0001

Low Nitrogen-High Phosphorus (nP) 40 6.2 0.4 1.98 bc n/a n/a 0.21 1.18 < 0.0001

Nutrient (Medium) 280 n/a n/a n/a 2.05 2.35 n/a n/a < 0.0001

Control 40 9.9 0.8 4.19 a n/a n/a 0.23 1.90 n/a

High Nitrogen (HN) 40 4.8 0.3 2.31 b n/a n/a 0.23 1.60 < 0.0001

Low Nitrogen (LN) 40 4.7 0.3 2.02 b n/a n/a 0.23 1.07 < 0.0001

High Phosphorus (HP) 40 4.7 0.4 2.11 b n/a n/a 0.23 1.62 < 0.0001

Low Phosphorus (LP) 40 3.2 0.3 2.15 b n/a n/a 0.23 1.20 < 0.0001

High Nitrogen-Low Phosphorus (Np) 40 7.2 0.3 2.15 b n/a n/a 0.23 1.57 < 0.0001

Low Nitrogen-High Phosphorus (nP) 40 6.2 0.4 1.54 b n/a n/a 0.23 0.92 < 0.0001

Nutrient (High) 280 n/a n/a n/a 1.97 2.28 n/a n/a < 0.0001

Control 40 9.9 0.8 4.19 a n/a n/a 0.20 1.90 n/a

High Nitrogen (HN) 40 3.8 0.3 2.02 bc n/a n/a 0.20 0.90 < 0.0001

Low Nitrogen (LN) 40 4.7 0.3 1.84 c n/a n/a 0.20 1.10 < 0.0001

High Phosphorus (HP) 40 3.5 0.2 1.32 c n/a n/a 0.20 0.72 < 0.0001

Low Phosphorus (LP) 40 6.8 0.1 2.82 b n/a n/a 0.20 1.65 < 0.0001

High Nitrogen-Low Phosphorus (Np) 40 5.2 0.2 1.92 c n/a n/a 0.20 1.17 < 0.0001

Low Nitrogen-High Phosphorus (nP) 40 4.8 0.4 1.88 c n/a n/a 0.20 0.95 < 0.0001

Atrazine 160 n/a n/a n/a 1.93 2.06 n/a n/a < 0.0001

Control 40 9.9 0.8 4.19 a n/a n/a 0.21 1.90 n/a

Low 40 7.2 0.3 1.96 b n/a n/a 0.09 1.23 < 0.0001

Medium 40 7.4 0.1 2.05 b n/a n/a 0.09 1.37 < 0.0001

High 40 6.8 0.1 1.85 b n/a n/a 0.09 1.19 < 0.0001

Atrazine (HN) 160 n/a n/a n/a 2.06 2.60 n/a n/a < 0.0001

Control 40 9.9 0.8 4.19 a n/a n/a 0.22 1.90 n/a

Low 40 4.8 0.3 1.87 b n/a n/a 0.22 1.02 < 0.0001

Medium 40 6.6 0.2 2.31 b n/a n/a 0.22 1.60 < 0.0001

High 40 3.8 0.3 2.02 b n/a n/a 0.22 0.90 < 0.0001

Atrazine (LN) 160 n/a n/a n/a 1.76 2.36 n/a n/a < 0.0001

Control 40 9.9 0.8 4.19 a n/a n/a 0.21 1.90 n/a

Low 40 4.7 0.3 1.41 b n/a n/a 0.21 0.89 < 0.0001

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Table 1 (continued)

Source n Max Min Mean Group Mean Grand Mean SE SD p value

Medium 40 5.1 0.3 2.02 b n/a n/a 0.21 1.07 < 0.0001

High 40 4.7 0.3 1.84 b n/a n/a 0.21 1.10 < 0.0001

Atrazine (HP) 160 n/a n/a n/a 1.69 2.31 n/a n/a < 0.0001

Control 40 9.9 0.8 4.19 a n/a n/a 0.22 1.90 n/a

Low 40 4.7 0.4 1.64 bc n/a n/a 0.22 0.80 < 0.0001

Medium 40 6.8 0.2 2.11 b n/a n/a 0.22 1.62 < 0.0001

High 40 3.5 0.2 1.32 c n/a n/a 0.22 0.72 < 0.0001

Atrazine (LP) 160 n/a n/a n/a 2.21 2.70 n/a n/a < 0.0001

Control 40 9.9 0.8 4.19 a n/a n/a 0.23 1.90 n/a

Low 40 3.2 0.3 1.66 b n/a n/a 0.23 0.80 < 0.0001

Medium 40 5.1 0.5 2.15 bc n/a n/a 0.23 1.20 < 0.0001

High 40 6.8 0.1 2.82 c n/a n/a 0.23 1.65 0.0002

Atrazine (Np) 160 n/a n/a n/a 2.24 2.69 n/a n/a < 0.0001

Control 40 9.9 0.8 4.19 a n/a n/a 0.27 1.90 n/a

Low 40 7.2 0.3 2.52 b n/a n/a 0.27 1.99 0.0001

Medium 40 7.4 0.1 2.15 b n/a n/a 0.27 1.57 < 0.0001

High 40 5.2 0.2 1.92 b n/a n/a 0.27 1.17 < 0.0001

Atrazine (nP) 160 n/a n/a n/a 1.76 2.40 n/a n/a < 0.0001

Control 40 9.9 0.8 4.19 a n/a n/a 0.21 1.90 n/a

Low 40 6.2 0.4 1.98 b n/a n/a 0.21 1.17 < 0.0001

Medium 40 4.4 0.2 1.54 b n/a n/a 0.21 0.92 < 0.0001

High 40 4.8 0.4 1.88 b n/a n/a 0.21 0.95 < 0.0001

Statistical significance among the tensile root strength means of the levels in each treatment is denoted by different letter superscripts (p < 0.05).Statistical significance between each treatment level and Control is indicated by p values <0.05

Table 2 Summary of one-wayWelch’s ANOVA tests of the ten-sile root strength response vari-able for the atrazine treatment andnutrient addition main effects, andmain effect subsets (in parenthe-ses) used to test for interactiveeffects

Source 1DFNum 2DFDen F Ratio p value

Nutrient 6 266.8 12.6 < 0.0001

Nutrient (High) 6 108.2 15.9 < 0.0001

Nutrient (Medium) 6 107.3 16.4 < 0.0001

Nutrient (Low) 6 107.7 17.9 < 0.0001

Atrazine 3 165.7 18.9 < 0.0001

Atrazine (HN) 3 83.8 16.3 < 0.0001

Atrazine (LN) 3 84.9 23.1 < 0.0001

Atrazine (HP) 3 82.3 27.0 < 0.0001

Atrazine (LP) 3 82.1 22.2 < 0.0001

Atrazine (Np) 3 84.7 14.2 < 0.0001

Atrazine (nP) 3 84.6 20.8 < 0.0001

Statistical significance is indicated by p < 0.051Degrees of Freedom –Numerator2 Degrees of Freedom - Denominator

772 Wetlands (2019) 39:759–775

Table 4 Results of nutrient tissue content testing of live S. patens above- (stem) and belowground biomass (roots) for carbon, nitrogen, and phosphorusas well as carbon-nitrogen (C:N) and nitrogen-phosphorus (N:P) ratios

Treatment Carbon (mmol/g) Nitrogen (mmol/g) Phosphorus (mmol/g) C:N N:P

Roots Stem Roots Stem Roots Stem Roots Stem Roots Stem

HN 36323a 35997ac 468.8c 376.1de 48.3a 39.1ac 77.5 95.7 9.7 9.6

LN 36023a 36261ac 500.6bc 359.3d 59.4b 37.5ac 72.0 100.9 8.4 9.6

HP 36134a 37033d 473.3c 387.3de 65.3b 39.1ac 76.4 95.6 7.2 9.9

LP 36393a 37164d 553.1ab 381.0de 61.2b 36.5ac 65.8 97.5 9.0 10.4

Np 35573b 36456cd 524.8abc 405.3ef 61.9b 37.3ac 67.8 89.9 8.5 10.9

nP 34656b 37069d 580.0a 431.8cf 59.4b 38.3ac 59.8 85.8 9.8 11.3

Control 36237ac 36546ac 454.1c 363.3d 43.0a 32.3a 79.8 100.6 10.6 11.2

Mean values with different letter subscripts are significantly different (p < 0.05). Comparisons of the means were made within each nutrient betweentreatments and control as well as between roots and stems

Table 3 Summary of soilparameters of a nutrient-atrazineinteraction experiment delineatedby atrazine treatment

Parameter Experimental Units

Low Medium High Control

Soil Temperature (°C)

Mean 26.6a 26.3a 26.3a 26.1a

Min 19.7 19.8 19.8 19.9

Max 31.4 31.2 31.3 31.0

Standard Error 0.45 0.40 0.41 0.38

pH

Mean 7.1a 7.1a 7.1a 7.1a

Min 7.0 7.0 7.0 6.9

Max 7.4 7.3 7.3 7.6

Standard Error 0.02 0.01 0.01 0.03

Redox Potential (mV)

Mean −12.3a −14.0a −14.4a −17.5a

Min −26.6 −22.2 −23.3 −30.1Max −3.6 −7.3 −7.8 −4.8Standard Error 0.8 0.6 0.6 1.1

Mean values with different letter subscripts are significantly different (p < 0.05)

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